Gaucher Disease: We extended our basic investigations on the pathogenesis of the neuronopathic forms of Gaucher disease. In addition to augmented glucocerebroside, there are markedly increased quantities of glucosylsphingosine (GlcSph) in the brain of these patients. GlcSph is neurotoxic and contributes significantly to the dysfunction and destruction of brain cells. We have identified six compounds that inhibit the enzymatic synthesis of GlcSph and ranked them in the order of their effectiveness. We shall determine the ability of such inhibitors to cross the blood-brain barrier. Using compounds that reach the brain in an effective and non-toxic concentration, we propose to conduct clinical trials with these agents to improve the debilitating clinical course in patients with neuronopathic Gaucher disease. We are investigating genomic changes that occur in Gaucher disease. We have identified 203 genes whose expression is increased 10-fold or more over normal and 78 genes whose function is decreased 10-fold or more. These findings are very highly statistically significant. We anticipate that this genomic analysis will provide considerable understanding with regard to differences in the phenotypes of patients with this disorder. For example, the expression of several well-known genes involved in cell division is greatly increased and may play a major role in the genesis of the hepatosplenomegaly that occurs in patients with Gaucher disease. We are exploring strategies to extend the organ and tissue distribution of exogenous glucocerebrosidase (GC) to augment the effectiveness of enzyme replacement therapy for patients with Gaucher disease. These efforts include the utilization of intracellular transport and membrane recognition domains linked to GC. We prepared recombinant GC that containing an in-frame fusion to the HIV-1 trans-activator protein transduction domain (TAT). Recombinant GC-TAT was expressed in eukaryotic cells from which catalytically active, normally glycosylated GC-TAT fusion protein was obtained. GC-TAT was taken up by cells such as fibroblasts which lack the mannose lectin on their surface that is utilized to target exogenous glucocererbrosidase to lipid-storing macrophages. It is expected that GC-TAT will be more efficiently delivered than unmodified GC to cells in the bone marrow and lung, and perhaps additional cells that lack the mannose lectin and thereby enhance the clinical responses of patients with Gaucher to enzyme replacement therapy. In collaboration with members of the Surgical Neurology Branch (SNB), we carried out an investigation of the safety of intracerebral injection of GC in non-human primates using the convection-enhanced delivery (CED) technique developed by SNB. Recipient animals showed no toxicity or immunological reaction to GC administered in this fashion. It has been shown previously that systemic GC alone did not alter the rapid progression of brain damage in patients with type 2 Gaucher disease because intravenously injected GC does not reach the central nervous system due to the blood-brain barrier. We have prepared and submitted clinical protocols to explore intracerebral administration of GC in patients with type 2 (severe neuronopathic) Gaucher disease who also receive GC systemically. Supplementation of intravenous with intracerebral GC appears essential to halt or reverse brain damage in patients with type 2 Gaucher disease. We are developing methods to improve gene therapy for patients with Gaucher disease using lentiviral vectors. The first of these constructs increased the level of glucocerebrosidase activity significantly in multiple organs and tissues when injected into experimental animals. Moreover, it very effectively transduces bone-marrow stem and progenitor cells ex vivo. We demonstrated long-term expression of GC in experimental animals following transplantation of bone-marrow-derived cells transformed by a GC lentivirus vector. It is anticipated that autologous cells transduced with this, or a related lentiviral vector, may be appropriate for gene therapy trials in patients with Gaucher disease since successful bone marrow transplantation can cure patients with type 1 (non-neuronopathic) Gaucher disease. In addition to our ongoing investigation of active-site specific molecular chaperone therapy in Fabry disease (v.i.), we shall examine the potential of this approach for the treatment of patients with Gaucher disease. This technique is based on the ability of certain compounds to interact with the catalytic site of mutated glucocerebrosidase and escort it from the endoplasmic reticulum where it is produced through the golgi apparatus to lysosomes. The low pH in lysosomes causes chaperones to dissociate from the enzyme and allows it to carry out its catalytic function. All surviving patients with Gaucher disease have residual glucocerebrosidase activity and are therefore potential candidates for molecular chaperone therapy. Fabry disease: We have identified a number of patients with this disorder in whom the reduced catalytic activity of alpha-galactosidase A is increased in the presence of the molecular chaperone 1-deoxy-galactonojirimycin (DGJ). We are examining the ability of DGJ to augment residual alpha-galactosidase A activity in cultured skin fibroblasts, T cells and peripheral blood mononuclear cells derived from patients with Fabry disease. A protocol has been approved for a Phase 1 safety and dose-response trial with DGJ that will be implemented in the near future. When it is completed, we shall examine the clinical effectiveness of active site-specific chaperone therapy in patients with Fabry disease with enhanceable alpha-galactosidase A activity (cf. lead investigator's report). Mucolipidosis IV (MLIV): The protein that is mutated in patients with this disorder is a member of the transient receptor potential protein family and is designated as MCOLN1. We prepared a targeted gene construct with an interruption of exon 1 of the MCOLN1 gene to attempt to reduce the formation of MCOLN1 and produce a knock-out murine model of MLIV. The mouse has a truncated transcript in the 5' coding region of MCOLN1. Expression of the modified MLIV protein is being characterized and detailed pathological examination of the organs and tissues of the mouse are underway. If this knock-out model does not exhibit a significant phenotype, we shall prepare other gene targeting constructs that interrupt additional portions of the MCOLN1 gene.